East African Journal of Veterinary and Animal Sciences (2018) Volume 2 (1): 1-10

Prevalence of Bovine Mastitis, Risk Factors and major Causative Agents in , East

Ketema Boggale, Bekele Birru*, Kifle Nigusu, and Shimalis Asras

Hirna Regional Veterinary Laboratory, P.O. Box 36, Regional State, Ethiopia

Abstract: A cross-sectional study was conducted from October 2009 to March 2010 in , , , Mieso and districts to estimate the prevalence of bovine mastitis, identify associated risk factors and isolate the predominant bacterial agents. A total of 1019 lactating cows were examined clinically and using California Mastitis Test (CMT) for subclinical mastitis. Standard bacteriological techniques were employed to isolate the bacterial agents. A total of 393 (38.6%) cows were positive for both clinical and subclinical mastitis. Among the risk factors, age, parity, and lactation stages were not significantly (p>0.05) associated with prevalence of mastitis. However, hygienic status of animals was associated with the occurrence of mastitis (p<0.05). Both contagious and environmental bacteria were isolated from milk samples collected from mastitic cows. The predominant bacteria isolate was Staphylococcus aureus with proportion of 14.1% followed by S. agalactiae with isolation proportion of 13.2%. E. coli and S. intermedius were the third predominant isolates with a rate of 10.9% for each. Among the seven antibiotics tested, gentamycin, amoxicillin, oxytetracycline, ampicillin, and cloxacillin were effective, whereas streptomycin and penicillin showed poor efficacy. The study revealed that mastitis is significant problem of dairy cows in the study areas. Hence, awareness creation among dairy farmers should be made about the impact of the disease; and training on hygienic milking practice and treating of subclinically infected cows should be given.

Keywords: California mastitis test, Clinical mastitis, Risk factors, Subclinical mastitis

Introduction (Mungube et al., 2004). Mastitis as a disease, especially Ethiopia has the largest livestock population compared the subclinical form, has received little attention in the to other African countries and cows represent the country and efforts have only been concentrated on the largest population of cattle of the country (CSA, 2009). treatment of clinical cases (Girma, 2001). The Milk produced from these animals provides an pathogens responsible for causing mastitis are broadly important dietary source for the majority of rural as classified into contagious and environmental agents well as considerable number of the urban and peri- (Quinn et al., 2002). The common microorganisms urban population. However, milk production often responsible for mastitis and spoilage of milk includes does not satisfy the country’s population requirement. Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium According to the reports of FAO (2015a & b), the total bovis, Mycoplasma species, and Streptococcus uberis (Erskine, annual national raw milk production in Ethiopia ranges 2001), coliforms (Escherichia coli, Klebsiella species and from 797,900 to 1,197,500 metric tons equivalents of Enterobacter aerogenes), Serratia, Pseudomonas, Proteus which 85 to 89 % is contributed by cattle. However, species, environmental Streptococci, and Enterobacter this amount is by far below the national demand for species (Quinn et al., 2002). milk and milk products, showing the considerable In Ethiopia, the available information indicates that potential for increasing small holder income and bovine mastitis is one of the most frequently employment opportunities across the dairy production encountered and prevalent diseases of dairy cows value chain. causing economic loss as a result of reduced milk yield, Mastitis is among important health problems in dairy discarded milk, and early culling of productive cows cattle and considered as one of the most important (Fekadu, 1995; Mekonnen et al., 2005). The reported threats affecting the dairy industry throughout the prevalence of mastitis in different parts of Ethiopia country (Biffa et al., 2005). Mastitis is an inflammation ranges from 1.2% to 21.5% (Kassa et al., 1999; Lemma of the mammary gland and udder tissue that usually et al., 2001; Mungube, 2001; Workineh et al., 2002; occurs as an immune response to bacterial invasion of Kerro and Tareke, 2003). the teat canal by variety of bacterial sources present on Across the globe, various reports indicated that S. the farm, and can also occur as a result of chemical, aureus showed resistance to penicillin. Previous study in mechanical and thermal injury to the udder (Wellenberg Italy (Gentilini et al., 2000), Argentina (Gianneechini et et al., 2002; Radostits et al., 2007). It is mostly a result of al., 2002), and Uruguay (Pitkala et al., 2004) reported a combined interplay among exposure to microbes, cow resistance rate of 47%, 40%, and 47%, respectively. In defence mechanisms and environmental risk factors Ethiopia, S. aureus isolates were highly resistant to (Suriyasathaporn et al., 2000). Mastitis is a management clindamycin (73.8%), bacitracin (72.1%) and related disease whose prevention and control depends vancomycin (70.5%) (Shimelis, 2014). The high among other factors on good management practices incidence of resistant bacteria might be due to ______*Corresponding Author. E-mail: [email protected] ©Haramaya University, 2018 ISSN 2616-8804 (Print) Ketema et al. East African Journal of Veterinary and Animal Sciences 2 (1): 1-10 indiscriminate use of antibiotics and intra-mammary years), adults (> 6 to 10 years), and old (> 10 years). preparations containing combinations or single broad- Parity was categorized as few (1- 3 calves), moderate (4- spectrum antibiotics (Pitkala et al., 2007). Edward et al. 7 calves), and many (> 7calves) (Berhanu et al., 2010). (2002) suggested a possible development of resistance Stage of lactation was categorized as early (1 up to 4 from prolonged and indiscriminate usage of some months), mid (4 up to 8 month), and late (8 month up antimicrobials. to the beginning of dry period) and hygiene of udder In west Hararghe Zone, famers reported mastitis as was categorized as Washing/drying, Washing only and one of the health problems among dairy cattle. not at all (Berhanu et al., 2010). In the study area, dairy However, to the best of our knowledge, a systematic cattle are managed under semi- intensive management study was not conducted to provide detailed system and provided natural pasture and agricultural by information on cattle mastitis. Thus, the study was product. conducted to estimate the prevalence of bovine mastitis, assess risk factors, isolate and identify the Study Design bacterial causative agents, and determine the drug A cross-sectional study was conducted from October resistance patters of the isolated bacteria in the study 2009 to March 2010 at cow and quarter level based on area. clinical and subclinical mastitis and indirect test (California mastitis test and culture) for subclinical Materials and Methods mastitis, microbial isolation and in-vitro antimicrobial Study Areas susceptibility test. The study was conducted in west Hararghe (latitude: 7°50’–9°50’ N; longitude: 40°00’–41°25’ E; altitude: Sampling and Sample Size Determination 1200–3060 m) administrative zones which are located For estimation of the prevalence of mastitis in the in the eastern part of Oromia Regional State and share study area, the sample size was determined by assuming boundaries with Afar Regional State, Somali Regional the expected prevalence to be 50% with the 95% State, as well as the . The livestock confidence level and desired precision of 5% using the population of west Hararghe is 1,149,089 cattle, formula described by Thrusfield (2005). Accordingly, 510,164 sheep and goats (WHZBA, 2006). It has three the minimum sample size required from each of the distinct agro-ecologies and different farming system five districts was 384 but only a total of 1019 cows that consists of highland (17.5%), mid highland were taken since the study was conducted in dry season (28.5%), and lowland (54.0%) and have two rainy in May and June and some of the areas were not seasons, the short rainy season and the main rainy accessible due to lack of transportation. The rural season, with a mean annual rainfall ranging from below kebeles, villages and lactating cows were also selected 700 mm in the lowlands to nearly 1200 mm at higher based on their accessibility to transportation. altitudes. Most of the people living in Hararghe lowlands are nomadic agro-pastoralists who move their Study Methodology livestock seasonally, following grazing opportunities Milk sampling procedure: History of the selected and water availability (Guinand, 2000). milking cow was taken to record information such as number of parturitions, parity, treatment, and physical Study Animals examination of the udder to determine blind teats and The study was conducted in districts that have major other defects and inflammation. The milk samples were towns with significant milk flow in the operational area taken from cows that are not treated with either intra- of Regional Veterinary Laboratory. Lactating mammary or systematic antimicrobials agents. The cows of local breed 15 days after parturition were udder was cleaned with warm water, antiseptics and selected for the study. The lactating cows were selected dried with clean towel. The first few drops of the milk purposively from three villages in each district and two squirt were discarded and milk from each quarter of the kebeles. The study populations were all local breeds of udder was taken into sterile bottles. The bottles were dairy cows that are managed under mixed crop- clearly labelled with an appropriate identification of the livestock farming system. Milk was collected from 17 cow’s number and quarter using permanent marker. rural kebeles. The study was conducted on 1019 local The samples were transported in ice box to the breed cows from the selected districts that include laboratory without delay and it were processed Boke, Darolebu, Kuni, Mieso, and Tulo, and 34, 130, immediately (Quinn et al., 2002). The screening test, 83, 513, and 259 cows, respectively were taken. The California mastitis test (CMT) was done at the field to number of sample cows vary among the districts diagnose the presence of subclinical mastitis according because the study was conducted in dry period and to the procedures of Quinn et al. (1999). Milk samples required number of cows were not available in some were poured into four shallow cups in the CMT paddle areas, and villages in some of the districts were not and equal amount of CMT reagent was added to each accessible due to lack of transportations. Age, parity, cup and gentle circular motion was applied to the lactation stage, and hygiene of cow’s udder were mixture on the horizontal plane. Based on the thickness explanatory variables used to associate with prevalence. of the gel formed by CMT reagent and milk mixture, Age of the cows was categorized as young (3 to 6 test results were scored as 0 (negative), 1 (weak

2 Ketema et al. Bovine Mastitis and its Major Causative Agents positive), 2 (distinct positive) and 3 (strong positive). disc was pressed down to ensure complete contact with Milk samples with CMT 1 to 3 were classified as the agar surface. Plates were inverted and incubated at evidence of subclinical mastitis (Quinn et al., 1999; 37°C for 24 hrs. The diameter of zone of inhibition Radostits et al., 2007). In the laboratory, samples were was measured in millimetres using calliper. After cultured immediately or stored at +4 °C in any case of measuring the zone of inhibition, it was classified as delay (NMC, 1999). Analysis of positive samples was susceptible, intermediate and resistant according to performed on isolation and identification of pathogenic National Committee for Clinical Laboratory Standard bacteria at Hirna regional veterinary laboratory of (NCCLS) break point to interpret the inhibition zone microbiology department. (Quinn et al., 2002).

Culture and isolation of bacteria: A loop full of the Data Management and Statistical Analysis milk sample was streaked on blood agar base enriched The data collected during sampling and laboratory with 7% sterile sheep blood and MacConkey agar and work were entered and stored in MS-excel. Before incubation was made for 24 to 48 hours at 37 oC. Plates subjected to statistical analysis, the data were were examined for the presence or absence of growth thoroughly screened for errors and properly coded. An on MacConkey agar, presence or absence of haemolytic intercooled Stata 9 software package (Stata characteristics on blood agar, size, shape, outline, Corporation, 2005) was used to perform the statistical elevation, consistence, and pigmentation. Sub culturing analysis. Descriptive statistical analysis such as was performed to obtain pure colony as well as for frequency and proportions were used to compute further examination of isolates on differential media. mastitis prevalence and antimicrobial susceptibility. Identification of bacterial species was done according Proportion of mastitis was calculated by dividing the to Quinn et al. (1999). The primary tests used for number of animals positive to the total sampled characterization of isolates were catalase, motility (semi animals. Pearson chi-square (χ²) test was employed to solid method), oxidise, and oxidation-fermentation assess the existence of association between mastitis and (OF), while secondary biochemical test was performed its risk factors. P-value <0.05 was considered using mannitol salt agar, triple sugar iron agar, indole, significant whereas P-value >0.05 was considered non- methyl red, Voges-Proskauer, citrate (IMViC), urease significant. and carbohydrate fermentation. Results In-vitro antimicrobial sensitivity test: Susceptibility Prevalence of Mastitis of bacteria to the commonly used antimicrobials was Among the total 1019 dairy cow examined, 393(38.6%) conducted using Kirby-Bauer disc diffusion method were detected positive for both clinical and subclinical (Quinn et al., 2002). Seven antimicrobial discs that mastitis. On physical examination of udder, 47/1019 include ampicillin, amoxicillin, penicillin, cloxacillin, (4.6%) of the cows revealed clinical mastitis while gentamycin, oxytetracycline, and streptomycin were 346/1019 (33.9%) showed subclinical mastitis. The selected from the main class of antimicrobials and used highest and lowest prevalence of mastitis were recorded for susceptibility testing. The bacteria culture was in Tulo district (42.47%) and Gamachis district streaked on Mueller-Hinton agar. Four up to five (32.5%), respectively (Table 1). antimicrobial discs were applied on the surface of each inoculated agar plates under aseptic conditions. Each

Table 1. Prevalence of clinical and subclinical mastitis at cow level in the study areas Type of mastitis Districts № of cows Total № of Over all Clinical form Subclinical form examined cows affected prevalence (%) № of positive Prevalence № of positive Prevalence (%) (%) Boke 34 12 35.29 3 6.38 9 2.6 Darolube 130 44 33.84 9 19.14 35 10.1 Gamachis 83 27 32.5 5 10.63 22 6.36 Mieso 513 200 38.98 19 40.42 181 52.3 Tulo 259 110 42.47 11 23.4 99 28.6 Total 1019 393 38.6 47 4.6 346 33.9

The overall prevalence of mastitis at quarter level was (58.3%) were negative while the remaining 211 (5.18%) 36.48% (1487/4076) of which 4% (163/1487) showed were blind (Table 3). Based on quarter location, the clinical signs such as fibrosis, cardinal signs of highest prevalence of mastitis was recorded at Right inflammation, visible injury, and tick infestation, front (9.4%) while the lowest was recorded in right atrophy of the tissue of udder and swelling of the hind (8.6%) (Table 3). supramammary lymph nodes (Table 2). However, 2378

3 Ketema et al. East African Journal of Veterinary and Animal Sciences 2 (1): 1-10

Risk Factors of Mastitis 14.09% followed by S. agalactiae (13.2%), and E. coli and The result showed that the prevalence of mastitis was S. intermedius with proportion of 10.9% for each. C. higher in adult animals (56.8%) followed by old age ulcerans was the least isolate which accounts for 1.7% group, and younger category. However, there was no (Table 5). statistically significant difference (p=0.208) among the age categories. Hygienic condition showed statistically Antimicrobial Susceptibility Profiles of Bacterial significant effect on the occurrence of mastitis Isolates (p<0.05). Thus, higher mastitis case was reported in From the total 1455 bacterial isolates, 972 were tested dairy cows managed under poor hygiene followed by for susceptibility to antimicrobials. The majority of those under intermediate hygienic condition (Table 4). isolated bacteria showed susceptibility to gentamycin (85.29%), amoxicillin (78.19%), oxytetracycline Prevalence of Bacterial Isolates (72.3%), ampicillin (67.18%) and cloxacillin (61.73 %), Out of 1487 mastitis positive quarters, 1455 harbours whereas streptomycin and penicillin showed poor bacteria belonging to 8 genera and 13 species. Thirty efficacy on most of the tested isolates. Cloxacillin and eight of the isolates were from samples with clinical streptomycin were effective against Corynebacterium and mastitis while the remaining 1417 isolates were from A. pyogenes, while Staphylococcus, Streptococcus, P. multocida, samples with subclinical mastitis. Both contagious and and the Enterobacteriaceae were highly susceptible to environmental bacteria were isolated. The predominant gentamycin, amoxicillin, oxytetracycline, ampicillin, and bacteria isolated were S. aureus with proportion of cloxacillin (Table 6).

Table 2. Prevalence of clinical and subclinical mastitis at cow and quarter levels Cow level (n=1019) Quarter level (n=4076) Types of mastitis № of positive Prevalence (%) № of positive Prevalence (%) Clinical 47 4.6 163 4 Subclinical 346 33.95 1324 32.48 Total 393 38.6 1487 36.48

Table 3. Occurrence of mastitis in cattle at the quarter level in five selected districts Blind quarter Negative quarter Positive quarter Quarter Total № № Prevalence (%) № Prevalence (%) № Prevalence (%) examined RF 1019 35 0.9 603 14.8 381 9.4 RH 1019 69 1.7 599 14.7 351 8.6 LF 1019 28 0.7 611 14.99 380 9.3 LH 1019 79 1.94 565 13.9 375 9.2 Over all 4076 211 5.18 2378 58.3 1487 36.48 RF= Right front; RH= Right hind; LF= Left front; LH= Left hind.

Table 4. Risk factors of mastitis in the study areas Variables Categories № examined № positive Prevalence (%) P-value χ2 CI Age group 3-6years (young) 573 142 24.8 6-9 years (adult) 431 245 56.8 0.208 1.149 0.020-0.092 >9 years (old) 15 6 40 Parity 1-3 calves (few) 551 175 31.8 4-7 calves (moderate) 379 141 37.2 0.094 3.574 0.085-0.006 >7 calves (many) 89 39 43.8 Lactation 1-4 month 137 76 55.47 stage 4-8 month 211 105 49.8 0.772 0.1751 0.040-0.030 8-10 month 578 199 34.4 Greater than 10 93 13 13.98 Udder hygiene Not at all 637 235 36.89 Washing 281 87 30.96 0.013 8.1713 0.1007-0.0117 Washing/drying 101 25 24.8

4 Ketema et al. Bovine Mastitis and its Major Causative Agents Table 5. Isolated mastitis causing microorganisms in the study areas Mastitis form Total Pathogens Clinical % Subclinical % № identified Prevalence (%) S. aureus 10 26.3 195 11.5 205 14.09 S. agalactiae 2 5.26 190 11.2 192 13.2 S. intermedius 2 5.26 156 11 158 10.9 E. coli 3 7.9 155 11 158 10.9 S. epidermidis 2 5.26 149 10.5 151 10.38 Klebsiella spp. 1 2.6 146 10.3 147 10.1 S. hyicus 3 7.9 108 7.6 111 7.63 A. pyogenes 3 7.9 86 6.06 89 6.1 C. bovis 3 7.9 60 4.2 63 4.3 Proteus spp. 4 10.5 67 4.7 71 4.9 P. multocida 1 2.6 46 3.24 47 3.2 S. faecalis 3 7.9 35 2.47 38 2.6 C. ulcerans 1 2.6 24 1.73 25 1.7 Over all 38 100 1417 100 1455 100

Discussion Ethiopia (Workineh et al., 2002; Kerro and Tareke, The present study showed an overall mastitis 2003; Mungube et al., 2004) and elsewhere in Africa prevalence of 38.6% based on CMT and clinical (Kivaria et al., 2004). Since environmental factors play examinations of the udder. This finding is comparable significant role, the prevalence of subclinical mastitis with the result reported by Workineh et al. (2002) and varies in dairy animals (Radostits et al., 2007). In Nesru (1999) who reported prevalence of 38.2% and Ethiopia, the subclinical form of mastitis received little 37.2% in Adami Tulu and urban and peri-urban dairy attention and efforts have been concentrated on the farms of Addis Ababa, respectively. In the country treatment of clinical cases, while high economic loss prevalence rate lower than the present finding were could also be resulted from subclinical mastitis (Biffa et reported by Hunderra et al. (2005) and Gizat (2004) in al., 2005; Hunderra et al., 2005). According to Erskine Sebeta (16.11%) and Bahir Dar (3.9%), respectively. (2001) the variation in prevalence between subclinical Karimuribo et al. (2006) from Tanzania reported lower and clinical mastitis may be due to the fact that the prevalence (14.2%) than the present finding. However, defence mechanism of the udder reduces the severity it is relatively lower than that obtained by Mungube et of the disease. Usually Ethiopian farmer’s, especially al. (2004) in central highlands of Ethiopia (46.6%). smallholders, are not well informed about the invisible Mastitis is a complex disease involving interactions of loss from subclinical mastitis since dairying is mostly a various factors such as management and husbandry side line business. This also apply to the present study practices, environmental conditions, animal risk factors, area where dairy farm owners do not screened their and causative agents, hence its prevalence will vary in cows for subclinical mastitis except seeking time and space (Radostits et al., 2007). veterinarian's assistance at times of clinical mastitis The prevalence of subclinical mastitis (33.95%) and occurrence. In the present study area, all farmers said clinical mastitis (4.6%) is comparable with the reports they do not practice washing/drying of udder. of Nesru (1999) (32.2% and 5%) and Bishi (1998) Moreover, their knowledge about mastitis was only on (30.2% and 5.5%) in urban and peri-urban dairy farms clinical mastitis and all of them do not have any at Addis Ababa. The relatively higher prevalence of information about subclinical mastitis, and surprised subclinical mastitis than that of clinical mastitis is in during the field testing seeing the CMT positive milk line with previous reports from different parts of reaction.

5 Ketema et al. East African Journal of Veterinary and Animal Sciences 2 (1): 1-10

Table 6. Antimicrobial susceptibility profiles of bacterial isolates Susceptibility to: Bacteria species Streptomycin Gentamycin Oxytetracycline Ampicillin Amoxicillin Penicillin Cloxacillin S I R S I R S I R S I R S I R S I R S I R S. aureus (n=205) 49.1 18.9 32.1 84.9 0.0 15.1 73.0 8.2 18.9 66.0 6.3 21.4 79.9 1.9 18.2 44.7 33.3 28.3 57.2 30.2 12.6 S. agalactiae (n=192) 58.1 18.4 23.5 84.6 6.6 8.8 74.3 11.0 14.7 72.8 7.4 19.9 78.7 11.8 9.6 53.7 27.9 18.4 64.0 23.5 12.5 S. intermedius (n=158) 63.2 13.2 23.7 88.6 8.8 2.6 76.3 10.5 13.2 73.7 9.6 16.7 83.3 7.9 8.8 57.0 8.8 34.2 69.3 11.4 19.3 E. coli (n=158) 57.8 19.3 22.9 87.2 8.3 4.6 74.3 11.9 13.8 70.6 11.0 18.3 78.0 13.8 8.3 50.5 22.0 27.5 63.3 18.3 18.3 S. epidermidis (n=151) 55.1 23.4 21.5 86.9 3.7 9.3 73.8 10.3 15.9 68.2 10.3 21.5 76.6 9.3 14.0 45.8 26.2 28.0 60.7 23.4 15.9 Klebsiella spp. (n=147) 60.4 16.5 23.1 91.2 0.0 8.8 78.0 12.1 9.9 71.4 11.0 17.6 82.4 11.0 6.6 49.5 11.0 39.6 65.9 22.0 12.1 S. hyicus (n=111) 63.9 8.3 27.8 86.1 2.8 11.1 76.4 9.7 13.9 70.8 13.9 15.3 81.9 0.0 18.1 55.6 36.1 31.9 69.4 9.7 20.8 A. pyogenes (n=89) 81.6 14.3 4.1 79.6 16.3 4.1 63.3 20.4 16.3 59.2 20.4 20.4 71.4 20.4 8.2 51.0 20.4 28.6 57.1 22.4 20.4 C. bovis (n=63) 54.3 8.6 37.1 77.1 0.0 22.9 65.7 5.7 28.6 57.1 8.6 34.3 71.4 2.9 25.7 45.7 8.6 45.7 85.7 8.6 5.7 Proteus spp. (n=71) 38.5 23.1 38.5 76.9 5.1 17.9 64.1 7.7 28.2 56.4 10.3 33.3 71.8 7.7 20.5 23.1 25.6 51.3 48.7 23.1 28.2 P. multocida (n=47) 28.0 24.0 48.0 84.0 0.0 16.0 56.0 8.0 36.0 48.0 12.0 40.0 72.0 0.0 28.0 16.0 16.0 68.0 40.0 32.0 28.0 S. faecalis (n=38) 38.1 9.5 52.4 81.0 19.0 0.0 61.9 14.3 23.8 52.4 19.0 28.6 71.4 14.3 14.3 23.8 0.0 76.2 42.9 14.3 42.9 C. ulcerans (n=25) 13.3 26.7 60.0 73.3 26.7 0.0 46.7 33.3 20.0 33.3 40.0 26.7 60.0 26.7 13.3 6.7 13.3 80.0 20.0 46.7 33.3 Values are in %; n= number of isolates tested; I= Intermediate; R= Resistant; S= Susceptible.

6 Ketema et al. Bovine Mastitis and its Major Causative Agents The overall quarter level prevalence recorded in the the contributing factors to transmit the bacteria from current study (36.48%) is comparable with the finding infected cows to healthy ones. of Nesru et al. (1999) and Abdelrahim et al. (1990) who In the present study, bacteria were isolated from the reported 37% and 39%, respectively. The quarter majority (1455/1487; 97.8%) of mastitis positive milk infection rate was higher than that reported by Kerro samples. The values recoded for the two predominant and Tareke (2003) (19%) in southern Ethiopia and organisms isolated from mastitis milk, S. aureus Biffa et al. (2005) (28.2%) in the country. The present (14.09%) and S. agalactiae (13.2%) are lower than study revealed that the front quarters are more exposed reported by earlier studies (Geresu, 1989; Tesfahiwot, to mastitis infection as compared to the hind quarters, 1996; Nesru et al., 1999) from Ethiopia. On the other which could be due to the fact that the front quarter hand, Tolassa (1987) and Mekuria (1986) reported far are highly predisposed for contamination with dirt. In higher prevalence of Streptococcus species (53.55 and addition to this, large amount of milk is produced from 45.50%). The predominance of these bacterial species front quarters and as a result the pressure on the teat is due to frequent colonization of teats as they are canal forces the canals to be opened widely and commonly found on the skin. The bacteria can easily allowing easy entrance of microbes. The observation of get access to the teat canal during milking or suckling blind quarters in this study might be an indication of a and can be transmitted from quarter to quarter and serious mastitis problem at the study districts. from cows to cows during milking practices. Their In the present study, prevalence of mastitis was ability to exist intracellular and localized within micro- numerically higher (P=0.208) in the adult (56.8%) and abscessation in the udder gave them chance to resist old (40%) age group than the young (24.8%) age group. antibiotic treatment (MacDonald, 1997), which could Kerro and Tareke (2003) and Busato et al. (2000) noted also be an important factor contributing to the that mastitis increase with increasing age since the predominance of these organisms. Kerro and Tareke lower immunity status of older animals predispose to (2003) found the coliform to account for about 20.96% mastitis, which is also confirmed by the result of the of the isolates and were the third predominant present experiment. The low mastitis prevalence in pathogens from dairy cows in Southern Ethiopia. cows with single parity and higher prevalence in cows Mekuria (1986) and Biffa (1994) reported lower with multiple parities is in accordance with previous percentage (3.64 and 3.14%) of E. coli than the present findings (Sergant et al., 1998; Busato et al., 2000; Kerro study. The prevalence of environmental E. coli may be and Tareke, 2003). Several factors can be involved in associated with poor farm cleanliness. Faeces which are the development of mastitis in animals with multiple common sources of E. coli can contaminate the parities. Increased clinical and subclinical mastitis in premises directly or indirectly through bedding, calving older cows increase the infection rates and decreases stalls, udder cleaning water and milker's hands the local defence mechanisms. (Radostits et al., 2007). The prevalence rate of other The tendency of high prevalence of mastitis at early isolates in the present study somewhat agreed with the stage of lactation as compared to mid and late lactation findings of Hamir et al. (1978), who reported 1.3%. Of stages agree with that reported by Kerro and Tareke all the isolates; contagious pathogens showed greater (2003) and Thennarasu and Muralidharium (2004) who frequency than others. In general, the prevalence of attributed this to the absence of dry period therapy and mastitis causing agents was high. birth related influences. Radostits et al. (2007) suggested The average susceptibility (74.4%) of S. aureus strains that the mammary gland is more susceptible to new to all antimicrobials tested is higher than susceptibility infection during the early and late dry period due to the reports of 62.7% reported by Mekonnen et al. (2005) in absence of udder washing and teat dipping, which in Ethiopia and Myllys et al. (1998) in Finland. However, turn may have increased the presence of potential Staphylococcus and Streptococcus species showed the pathogens on the skin of the teat. The occurrence of existence of resistance to streptomycin and penicillin. more cases during early lactation stage may be due to The resistance of S. aureus to penicillin may be absence of dry cow therapy and birth related attributed to the production of beta lactamase, an influences. The amount of milk ejected is also higher enzyme that inactivates penicillin and closely related during early lactation periods and this cause increase in antibiotics. It is believed that around 50% of mastitis patency of the teats and decreased local defence causing S. aureus strains produces beta lactamase (Green factors. and Bradely, 2004). Moreover, these two antibiotics are Milking hygienic condition is one of the predisposing most widely used in many parts of Ethiopia since they factors in the distribution of mastitis. The present study are affordable antibiotics to farmers and are sometimes revealed mastitis prevalence was significantly (P=0.013) the only available antibiotics in many veterinary clinics. higher when milking was conducted under poor This wide use of these drugs and inappropriate hygienic condition (36.89%) than good milking hygiene administration could have contributed to the (24.8%). Kivaria et al. (2004) noted higher prevalence of development of resistance by the predominant bacterial mastitis in animals kept on farms with poor hygiene agents in the area. P. multocida are the most susceptible and attributed this to increased exposure and isolates to gentamycin, amoxicillin, oxytetracycline, transmission of pathogens during milking. Moreover, ampicillin, and cloxacillin, but resistant to streptomycin poor milking hygiene and milking equipment may be and penicillin. The present study has demonstrated the

7 Ketema et al. East African Journal of Veterinary and Animal Sciences 2 (1): 1-10 existence of resistance bacteria to commonly used subclinical mastitis in peri-urban and urban dairy antimicrobial agents and the results are in accordance production system in Addis Ababa Region, with reports from earlier studies in other countries Ethiopia, MSc Thesis, Addis Ababa University, Free (Gentilini, 2000; Edward et al., 2002) that suggested a University, Berlin, Germany. possible development of resistance from prolonged and Busato, A., Trachsel, P., Schallibaum, M. & Blum, J. W. indiscriminate usage of some antimicrobials. It is (2000). Udder health and risk factors for subclinical therefore, very important to implement a systemic mastitis in organic dairy farms in Switzerland. application of an in-vitro antibiotic susceptibility test Preventive Veterinary Medicine, 44: 205-220. prior to the use of antibiotics in both treatment and CSA (Central Statistical Authority) (2009). Report on prevention of intra-mammary infections. livestock and livestock characteristics; Volume II, Ethiopia, Addis Ababa, 2010. Conclusion Edward, M., Anna, K., Michal, K., Henryka, L. & The present study showed prevalence of 38.6% based Krystyna, K. (2002). Antimicrobial susceptibility of on CMT and clinical examinations of bovine mastitis. staphylococci isolated from mastitic cows. Bulletin of Hygienic conditions has a significant association the Veterinary Institute in Pulawy, 46: 289-294. (P<0.05) with mastitic cows in the study areas. The Erskine, R. J. (2001). Intramuscular administration of predominant bacterial isolates were S. aureus (14.09%), ceftiofur sodium versus intrammamary infusion of followed by S. agalactiae (13.2%) and E. coli and S. penicillin/ novobiocin for treatment of Streptococcus intermedius with proportion of 10.9% for each. agalactiae mastitis in dairy cows. Journal of the Gentamycin, amoxicillin, oxytetracycline, ampicillin, American Veterinary Medical Association, 208: 258-260. and cloxacillin showed relatively good efficacy and can FAO (2008a). Livestock sector, brief livestock be used in the study area for treatment of mastitis. population, sector analysis and policy branch. Since some of the trial discs especially streptomycin Rome, Italy, pp: 1-15. and penicillin showed low potency, special attention FAO (2008b). Milking, milk production hygiene and should be given to avoid development of antimicrobial udder health. Animal production and health paper, resistance. We recommend awareness creation among series 78. FAO, United Nations, Rome, Italy, pp: farmers on the impacts of subclinical mastitis for 68-72. effective mastitis control. Moreover, the udder of each Fekadu Kebede (1995). Survey on the prevalence of cow should be periodically checked for the timely bovine mastitis and the predominant causative treatment and prevention of the disease as well as agents in Chaffa valley. In: proceedings of the 9th selection of effective antimicrobials. conference of Ethiopian Veterinary Association, 1995, Addis Ababa, Ethiopia, pp: 101-111. Conflict of Interests Gentilini, E., Denamiel, G., Llarente, P., Godaly, S., The authors declare that they have no competing Rebuelto, M. & Degregorio, O. (2000). interests. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. Journal of Dairy Science, 83: 1224-1227. References Geresu Biru (1989). Major bacterial causes of bovine Abdelrahim, A. I., Shommein, A. M., Suliman, H. B. & mastitis and their sensitivity to common antibiotics. Shaddard, S. A. (1990). Prevalence of mastitis in Ethiopian Journal of Agricultural Science, 11: 45-54. imported Freisian cows in Sudan. Revue D'élevage et de Gianneechini, R., Concha, C., Rivero, R., Delucci, I. & Médecine Vétérinaire des Pays Tropicaux, 42: 512-514. Moreno López, J. (2002). Occurrence of clinical and Berhanu Mekibib, Furgasa Megersa, Fufa Abunna, subclinical mastitis in dairy herds in the West Bekele Megersa & Regassa Alemayehu (2010). Littoral Region in Uruguay. Acta Veterinaria Bovine mastitis: Prevalence, risk factors and major Scandinavica, 43 (4): 221-230. pathogens in dairy farms of Holeta town, central Girma Tefera (2001). Prevalence of mastitis at Alemaya Ethiopia. Veterinary World, 3 (9): 397-403. University dairy farm. Journal of Ethiopian Veterinary Biffa Demelash (1994). The study on the prevalence of Association, 5: 17-21. bovine mastitis in indigenous Zebu cattle and Jersey Gizat Alamaw (2004). A cross-sectional study of breeds in Wollaita Sodo, characterization and in bovine mastitis in and around Bahir-Dar and vitro drug sensitivity of the isolates, DVM Thesis, antibiotic resistance patterns of major pathogens, Addis Ababa University, Faculty of Veterinary MSc Thesis, Addis Ababa University, Debre-Zeit, Medicine, Ethiopia, pp: 71-78. Ethiopia. Biffa Demelash, Debela Etana & Beyene Fiqadu Green, M. & Bradely, A. (2004). Clinical Forum- (2005). Prevalence and risk factors of mastitis in Staphylococcus aureus mastitis in cattle. UK VET, 9: 4. lactating dairy cows in Southern Ethiopia. Guinand, Y. (2000). Hararghe agro-pastoralists face an International Journal of Application Research in Veterinary uncertain future: focus on livelihoods in selected Medicine, 3: 189-198. belg-dependent areas of East and West Hararghe. Bishi Abebe (1998). Cross-sectional study and UN-Emergencies Unit for Ethiopia, field mission longitudinal prospective study of bovine clinical and report, 14-21 March 2000, Addis Ababa, Ethiopia.

8 Ketema et al. Bovine Mastitis and its Major Causative Agents Hamir, A. N., Gehring, W. & Mohammed, S. L. (1978). Myllys, V., Asplund, K., Brofeldt, E., Hirvel f-Koski, The incidence of bovine mastitis in Kenya. Bulletin V., Honkanen-Buzalski, T., Juntilla, J., Myllykangas, of Animal Health and Production in Africa, 26: 55-61. O., Niskanen, M., Saloniemi, H., Sandholm, M. & Hunderra Sori, Ademe Zerihun & Sintayehu Abdicho Saranpaa, T. (1998). Bovine mastitis in Finland in (2005). Dairy cattle mastitis in and around Sebeta, 1998 and 1995- changes in prevalence and Ethiopia. International Journal of Applied Research and antimicrobial resistance. Acta Veterinaria Scandinavica, Veterinary Medicine, 3: 332-338. 39: 119-126. Karimuribo, E. D., Fitzpatrick, J. L., Bell, C. E., Swai, Nesru Hussein, Teshome Yehualashet & Getachew E. S., Kambarage, D. M., Ogden, N. H., Bryant, M. Tilahun (1999). Prevalence of mastitis in cross-bred J. & French, N. P. (2006). Clinical and subclinical and Zebu cattle. Ethiopia Journal of Agricultural Science, mastitis in smallholder dairy farms in Tanzania: 16: 53. Risk, intervention and knowledge transfer. Preventive Nesru Hussien (1986). A survey of bovine mastitis Veterinary Medicine, 74: 84-8. around Sebeta, DVM Thesis, Faculty of Veterinary Kassa Tamir, Wirtu Gebrehawariat & Azage Tegegne Medicine, Addis Ababa University, Ethiopia. (1999). Survey of mastitis in dairy herds in the NMC (1999). Current Concept in bovine mastitis Ethiopian central highlands. Ethiopia Journal of National mastitis Council (NMC), 3rd ed., 1840 Science, 22: 291-301. Wilsonblud, Arlington, VA 22: 201. Kerro Dego & Tareke Fikadu (2003). Bovine mastitis Pitkala, A., Salmikivi, L., Bredbacka, P., Myllyniemi, A. in selected areas of Southern Ethiopia. Tropical L. & Koskinen, M. T. (2007). Comparison of tests Animal Health and Production, 35: 197-205. for detection of beta-lactamase-producing Kivaria, F. M., Noordhuizen, J. P. & Kapaja, A. M. Staphylococci. Journal of Clinical Microbiology, 45: (2004). Risk indicates associated with subclinical 2031-2033. mastitis in small holder dairy cows in Tanzania. Quinn, P. J., Carter, M. E., Markey, B. K. & Carter, G. Journal of Cell and Animal Biology, 3 (5): 071-077. R. (1999). Clinical veterinary microbiology, Mosby Lemma Megerssa, Kassa Tamir & Azage Tegegne International Limited, London, pp: 327-344. (2001). Clinically manifested major health problem Quinn, P. J., Carter, M. E., Markey, B. K. & Carter, G. of cross-breed dairy herds in urban and pre-urban R. (2002). Clinical veterinary microbiology, production system in the central highlands of Harcourt Publishers, Virginia, pp: 331-344. Ethiopia. Tropical Animal Health and Production, 33: Radostits, O. M., Gay, C. C., K. Hinchcliff, W. & 85-89. Constable, P. D. (2007). Veterinary Medicine: A MacDonald, U. S. (1997). Streptococcal and Text book of disease of cattle, sheep, pigs, goats, Staphylococcal mastitis. Journal of American Veterinary and horses, 10th ed., Ballier, Tindall, London, pp: Medical Association, 170: 1157. 674-762. Mekonnen Hilemariam, Shibeshi Workineh, Bayleyegne Sergant, J. M., Scatt, H. M., Lestie, K. E., Ireland, M. J. Mekonen, Moges Alemayehu & Tadele Kebede & Bashir, A. (1998). Clinical mastitis in dairy cattle (2005). Antimicrobial susceptibility profile of in Ontario, frequency of occurrence and bacterial mastitis isolates from the cows in three major isolates. Canadian Veterinary Journal, 34 (33): 240. Ethiopian dairies. Revue de Médecine Vétérinaire, 176 Shimels Tesfaye (2014). Isolation and antimicrobial (7): 391 394. susceptibility of Staphylococcus aureus and occurrence Mekuria Mengistu (1986). Prevalence and etiology of of methicillin resistant Staphylococcus aureus (MRSA) bovine mastitis in Bahir-Dar, DVM Thesis, Faculty in mastitic dairy cows in the Selale/Fitche area, of Veterinary Medicine, Addis Ababa University, North Showa, Ethiopia, MSc Thesis, College of Ethiopia. Veterinary Medicine and Agriculture, Addis Ababa Mungube, E. O. (2001). Management and economics University, Debre-Zeit, Ethiopia. of dairy cow mastitis in the urban and peri-urban Stata Corporation (2005). Stata statistical software. areas of Addis Ababa, MSc Thesis, Addis Ababa Release 9.0. College Station, Texas. University, Debre-Zeit, Ethiopia. Suriyasathaporn, W., Schukan, Y. H. N. & Nilsson, A. Mungube, E. O., Tenhagen, B. A., Kassa Tamir, (2000). Low somatic cell count, risk factors for Regassa Fufa, Kyule, M. N., Greiner, M. & subsequent clinical mastitis in dairy herds. Journal of Baumann, M. P. (2004). Risk factors for dairy cows’ Dairy Science, 83: 1248-1255. mastitis in the central highlands of Ethiopia. Tropical Tesfahiwot Zarihun (1996). Study on bovine subclinical Animal Health and Production, 36 (5): 463-472. mastitis at Stela Dairy farm, DVM Thesis, Faculty Mungube, E. O., Tenhagen, B. A., Regassa Fufa., of Veterinary Medicine, Addis Ababa University, Kuyle, M. N., Sheferaw Yimer, Kassa Tamir & Ethiopia. Baumann, M. P. O. (2005). Reduced milk Thennarasu, A. & Muralidharium, M. (2004). Effect of production in udder quarters with subclinical lactating parameters on the incidence of bovine mastitis and associated economic losses in cross mastitis. Indian Journal of Animal Sciences, 74 (4): 379- breed dairy cows in Ethiopia. Tropical Animal Health 381. and Production, 37 (6): 503-512. Thrusfield, M. (2005). Veterinary epidemiology, 3rd ed., Blackwell science Ltd., Oxford, pp: 232-234.

9 Ketema et al. East African Journal of Veterinary and Animal Sciences 2 (1): 1-10

Tolassa Abera (1987). A survey on bovine mastitis WHZBA (2006). West Hararghe Zone Bureaus of around Kallu province, DVM Thesis, Faculty of Agriculture. A technical report. Veterinary Medicine, Addis Ababa University, Workineh Shibeshi, Bayleyegn Mekonnen, Hailemariam Ethiopia. Mekonnen & Potgieter, L. N. D. (2002). Prevalence Wellenberg, G. J., Vanderpoel, H. M. & Vonoir, J. T. and etiology of mastitis in cows from two major (2002). Viral infection and bovine mastitis. Journal of Ethiopian dairies. Tropical Animal Health and Veterinary Microbiology, 88: 27-45. Production, 34 (1): 19-25.

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